We offer the following types of analyses: 1. Protein identification (from in-solution, 1D/2D gel) 2. Relative protein quantitation (label-free, SILAC, TMT, DIGE) 3. Peptide/protein fractionation using gels, offline basic reversed LC, IEF or SCX 4. Protein post-translational modifications identification and relative quantification (phosphorylation, acetylation, succinylation, malonylation, nitrosylation, ubiquitination, nitration, SUMO, sulfhydration) 5. Serum depletion and protein identification/quantification 6. We work with investigators on custom projects (either targeted proteins or systems biology approaches) In addition to helping the NHLBI investigators, our research involves developing new approaches for PTM characterization and absolute protein quantitation (e.g. measuring occupancy of nitrosylation with cys-TMT tags, acetylation occupancy, tissue ubiquitination, absolute quantification of a mitochondrial protein panel, protein cross-linking). Since 2013, we have supported over 60 investigators within NHLBI and outside, and have collected over $800,000 in user fees. We co-authored or were acknowledged in more than 60 papers since the last BSC and were a recipient of the Orloff Core Innovation Award in 2017 (For developing work to allow measurement of the occupancy of S-nitrosylation) and the NHLBI Group Award in 2017. Our individual research focuses on the following areas: 1. Nitrosylation identification and occupancy measurements (2017 Orloff Innovation Award and 2017 NHLBI Group Award): We developed a method using tandem mass tags by which Tish Murphys lab could measure the occupancy or mole fraction of S-nitrosylation, an important post-translational modification. Although post translational modifications are well described in the literature, there are very few methods that measure the occupancy of these modifications. We worked with Dr. Murphys lab to develop these methods for S-nitrosylation. This approach was published in Circulation Research: - Kohr MJ, Aponte A, Sun J, Gucek M, Steenbergen C, Murphy E. Measurement of S-Nitrosylation Occupancy in the Myocardium with Cysteine-Reactive Tandem Mass Tags. Circ Res. 111: 1308-1313, 2012; and - Sun J, Aponte AM, Menazza S, Gucek M, Steenbergen C, Murphy E. Additive cardioprotection by pharmacological postconditioning with hydrogen sulfide and nitric oxide donors in mouse heart: S-sulfhydration vs S-nitrosylation. Cardiovas. Res. 110:96-106, 2016.). Although many post-translational modifications have been identified, the biological impact of these modifications is hard to evaluate without methods to determine the percentage of a protein that contains the modification. The development of this method is key to understanding the biological significance of S-nitrosylation. 2. Protein cross-linking to identify interacting proteins: We are developing approaches to identify mitochondrial macromolecular complexes (with Dr. Balaban) and interactions within the HDL and LDL proteins (with Dr. Remaley). Combining cleavable cross-linkers such as DSSO with mass spectrometry (XL-MS) facilitates structural information from protein-protein interactions in complex systems. The cross-linking reagents contain an MS cleavable bond, which can fragment during collision induced dissociation (CID) prior to peptide backbone breakage. XL-MS workflow involves protein cross-linking (i.e. DSSO), trypsin digestion of cross-linked proteins, offline peptide fractionation (SCX) and LC/MSn analysis (Orbitrap Lumos) of resulting peptide mixtures. Currently, we are working on offline enrichment techniques to enrich the pool of crosslinked peptides, MS method development to improve the speed and accuracy for identifying complex crosslinked peptide mixtures and relative quantitation of cross-linked peptides using Tandem Mass Tags (TMT) to label up to 10 cross-link samples. We also developed an in-house software that easily identifies and collates crosslinks based on their MS3 spectra. 3. Proteogenomics: We are working with the Bioinformatics Core to develop a proteogenomic pipeline including experimental design, sample preparation, mass spectrometry data mining and the integration of MS data with next-generation sequencing data. In addition, we are building a sequence database consisting of known and unknown peptide sequences of long non-coding RNA (lncRNA) and long intergenic non-coding RNA (lincRNA) for researchers interested in clinical proteogenomics. 4. Characterization of methylation profiles for bacterial protein methyltransferases in Rickettsia: Methylation of rickettsial OmpB (outer membrane protein B) has been implicated in bacterial virulence. We used mass spectrometry to characterize methylation of recombinantly expressed fragments of Rickettsia typhi OmpB and native OmpBs purified from R. typhi and R. prowazekii strains. We found that in vitro trimethylation occurs at relatively specific locations in OmpB, whereas monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. These studies provide the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncovering the link between OmpB methylation and rickettsial virulence. - Abeykoon AH, et al. Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia. J Biol Chem. 2016 Sep 16;291(38):19962-74 and - Abeykoon A, et al. Multimethylation of Rickettsia OmpB catalyzed by lysine methyltransferases. J Biol Chem. 2014 Mar 14;289(11):7691-701) 5. Development of mitochondrial protein panel: We are developing targeted peptide analysis on Orbitrap Lumos to simultaneously follow specific mitochondrial proteins in healthy and diseased platelets (working with Drs. Balaban and Sack). 6. Serum proteomics: We are developing new approaches to comparatively analyze proteins in serum samples. With faster and more sensitive instruments, we can analyze the samples directly, i.e. without prior depletion of the most abundant proteins. Using undepleted serum samples increases the number of identified serum proteins and improves the variability in the sample preparation. In collaboration with Dr. Ackerman lab, we worked on a sickle cell anemia proteomics project where we compared 4 different groups (healthy, steady state, crisis, crisis recovery) to study the protein expression changes at different phases of the disease progression (27 patients total). We used TMT relative quantitation. The results will be compared with SOMA scan technology which uses aptamers to measure protein concentrations. In collaboration with Fitzhugh lab, we explored the effect of stem cell transplants on outcomes of sickle cell disease with 2 different groups (successful and unsuccessful transplant) in a 20-patient study. 7. Targeted clinical proteomics for fast clinical diagnosis of bacterial infections: With Suffredini lab, we developed a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification. We also developed a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides from Acinetobacter baumannii. We published several papers with Dr. Suffredinis group on fast clinical diagnosis using targeted proteomics: - Wang H et al. A Genoproteomic Approach to Detect Peptide Markers of Bacterial Respiratory Pathogens. Clin Chem. 2017 Jun 6. - Wang H et al. Peptide Markers for Rapid Detection of KPC Carbapenemase by LC-MS/MS. Sci Rep. 2017 May 31;7(1):2531